Our previous studies have demonstrated that mammalian cells have two distinct types of nucleoside transport that differ by 1000-fold in their sensitivity to the inhibitor nitrobenzylthionosine, and have defined the transport properties of model cell lines having one or the other (S49 mouse lymphoma and Walker 256 rat carcinosarcoma, respectively), or both (L1210 mouse leukemia) of these activities. The continuation of this project will focus on the purification of the proteins mediating NBMPR-sensitive and -resistant nucleoside transport, and comparison of the structural and functional properties of those proteins. Monoclonal antibodies will be obtained to use as specific probes of the transport proteins and to aid in their purification affinity chromatography utilizing NBMPR and adenosine as ligands will also be employed in the purification studies. The biological activity of the proteins will be monitored during purification by reconstitution of transport activity in lipid vesicles. Reconstitution will also be used to compare the functional properties of the purified proteins. The structures of the proteins will be compared by peptide mapping, and sequencing of the peptides bearing the substrate and NBMPR binding sites. The information gained in the study of the purified transport proteins will also be used to examine the structure and orientation of the proteins as they exist in their native state in the membranes of S49 and Walker 256 cells. The relationship between NBMPR-sensitive and - resistant transport will be examined in L1210 cells. These studies will use genetic and biochemical approaches to determine whether the two transport activities are properties of two separate and distinct proteins or two related forms of the same protein. The transport protein(s) of a cell line that exhibits anomolous NBMPR-binding properties will also be examined to gain a better understanding of the relationship between inhibitor binding sites and substrate permeation sites. It is expected that the information gained form these studies will contribute significantly to the long range goals of understanding the physiological roles of nucleoside transport in mammalian cells, and in designing approaches to using transport inhibitors to increase the selectivity of antimetabolites used in cancer chemotherapy.